Fluorescence is the most sensitive molecular detection and chemical imaging method available today. It is used to image even single molecules, in real time, with high spatial and spectral resolution, at ambient conditions, and with little perturbation. It also facilitates the detection and identification of pathogens, development of drugs, and other biomedical applications. Fluorescent dyes are commonly used to study intracellular chemical concentration changes, to measure immunochemical concentrations in a sample, to tag molecules on the surface of cells and in tissues, and in fundamental research on protein folding mechanisms. Nevertheless, typical background fluorescence from sample and instrument optics makes detecting and distinguishing low levels of fluorescence and small changes in fluorescence a challenging endeavor. Background fluorescence has hampered use of fluorescence measurement techniques in all areas and has fuelled development of red-exciting dyes, two photon excitation sources, chemiluminescent dyes, and innovative schemes and expensive equipment to bring background noise down to manageable levels.
It is an ultimate goal of medicine and biology to determine how cells function, and what effect drugs and other exogenous stimuli may have on them. Towards this goal, one must measure the chemical composition of cells in various conditions and environments (Taylor et al., Current Opinion in Biotechnology. 2001 February; 12(1):75-81). Chemicals of interest include pH that affects enzyme reactions; sodium, potassium, calcium, chloride ions and nitric oxide that are important in neuron signaling and osmosis; oxygen, carbon dioxide, ethanol, and glucose that are important in respiration; reactive oxygen species that are important for aging and photodynamic therapy; in addition physical characteristic such as temperature may also be measured. Biological macromolecules such as DNA, RNA, carbohydrates, and neurotransmitter proteins may also be detected. It is important to be able to detect multiple analytes at the same location at the same time in order to fully understand any physiological responses. It is also useful to detect at specific locations since analyte concentrations are often inhomogeneous.
In medical and biochemical research, when the domain of the sample is reduced to micrometer regimes, e.g. living cells or their subcompartments, the real-time measurement of chemical and physical parameters with high spatial resolution and negligible perturbation of the sample becomes extremely challenging. A traditional strength of chemical sensors (optical, electrochemical, etc.) is the minimization of chemical interference between sensor and sample, achieved with the use of inert, “biofriendly” matrices or interfaces. However, when it comes to penetrating individual live cells, even the introduction of a sub-micron sensor tip can cause biological damage and resultant biochemical consequences. In contrast, individual molecular probes (free sensing dyes) are physically small enough but usually suffer from chemical interference between probe and cellular components.
Perhaps the easiest way to measure cells' compositions is to grind up cells into a puree and analyze the puree's composition using electrochemical sensors, titration with indicator dyes, electrophoresis, or other means. However, blending the cells kills them, and makes it difficult to follow processes that may happen to living cells. In addition, grinding and blending the cells together makes it difficult to analyze different parts of a cell separately.
Microelectrodes and electrodes can measure chemical concentrations in living cells or in biological tissue, but they have a number of problems. Inserting the probe into the cell is invasive, and may kill the cell or affect how it functions. A reference electrode is required to make electrical measurements and proper placement and calibration of the reference electrode complicates the process. In addition, each microelectrode can only measure concentrations near the tip of the probe, so a single probe cannot show the spatial distribution of chemicals in a cell. In principle, multiple probes could be inserted to determine chemical concentrations at many points, however, each additional probe is successively more invasive, and most cells are too small to take more than one or two probes.
To form an image showing the distribution of chemical species in a cell, it is now a common practice to inject fluorescent indicator dyes into cells. The intensity, peak wavelength, polarization anisotropy, or lifetime of the dye fluorescence indicates chemical concentrations. Unfortunately, many fluorescent indicator dyes suffer from a number of problems. Many intensity indicator dyes (intensity is the most commonly used dye property) lack an internal reference so it is impossible to tell whether a strong intensity is due to a high analyte concentration or a large amount of dye.
Another problem with free dye is that the cellular environment can affect the fluorescence. Cellular proteins often quench dye fluorescence, affecting readings unpredictably. The dye may preferentially adhere to certain structures in a cell, making readings unrepresentative. In addition, the dye may be sequestered from the cell making readings for non-ratiometric probes change in time. Another problem with free dyes is that they may affect cell function or may poison the cell. Since each free dye interacts with a cell in its own way, the interaction needs to be studied for each type of dye and cell to ensure an accurate reading.
However, most fluorescent indicator dyes have broad excitation and emission peaks that limit the number of different dyes that can be detected without having some of the fluorescence from the different dyes overlap. This limits the number of analytes that can be detected independently at any given time.
Thus, improved methods for studying cells and intracellular analytes are needed. Such improved methods should be amenable to monitoring the cell at more than one location and should have minimal toxicity.